Calcium Triggered Compound Fusion at Ribbon Synapses During Exocytosis

Abstract

AbstractBackgroundExocytosis in photoreceptors involves fusion of vesicles with the plasma membrane at the base of a specialized proteinaceous electron‐dense structure known as a synaptic ribbon. The molecular mechanisms underlying exocytosis at ribbon synapses are not well understood. Research suggests a compound fusion model where vesicles at ribbon synapses undergo homotypic fusion forming larger vesicles prior to fusion with the plasma membrane. But how this process is mediated remains unclear. Ca2+ triggers several forms of exocytosis in different types of eukaryotic cells through activation of the SNARE complex. SNARE‐mediated fusion of membranes is triggered by the calcium sensor Synaptotagmin, but not much is known about whether this applies to compound fusion. Here, we report that, like typical synaptic exocytosis, compound fusion is mediated by Ca2+ influx.MethodTo examine the role of calcium in compound fusion, we performed vesicle purification experiments from Mus musculus (C57BL/6 WT) retina. Brain tissue was also used as a control where we expected no compound fusion. Retina and brain tissue were dissected, homogenized, and ultracentrifuged. The supernatant was incubated in 2mM Ca2+ for 5 minutes then vesicles from the Ca2+samples and controls containing no Ca2 were isolated using magnetic beads coupled with rho1D4 monoclonal antibody. Vesicles were negatively stained and viewed under TEM. Vesicle sizes of the Ca2+ and control samples were determined via blind analysis using ImageJ.ResultsIn retina samples, exogenous Ca2+ caused a significant increase in vesicle size when viewed under TEM. In the retina Ca2+ samples, vesicle diameter ranged from 30nm to 250nm. This effect was not seen in retina controls where vesicle diameter ranged from 30nm to 60nm. No large vesicles were seen in any of the brain samples.ConclusionOur research supports the compound fusion model of vesicle exocytosis at ribbon synapses. We suggest that in retina, the larger vesicles formed during exocytosis are a result of smaller vesicles fusing with each other and that Ca2+ has a significant role in this process. Future studies investigating the role of calcium sensors such as synaptotagmin will help to further elucidate the mechanisms underlying compound fusion and its therapeutic potential in neurological disorders.